American Association for Cancer Research
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Supplementary Figures 1 through 10 from Preserved Activity of CD20-Specific Chimeric Antigen Receptor–Expressing T Cells in the Presence of Rituximab

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journal contribution
posted on 2023-04-03, 23:04 authored by Gregory A. Rufener, Oliver W. Press, Philip Olsen, Sang Yun Lee, Michael C. Jensen, Ajay K. Gopal, Barbara Pender, Lihua E. Budde, Jeffrey K. Rossow, Damian J. Green, David G. Maloney, Stanley R. Riddell, Brian G. Till

Supplemental Figure S1. (A) Schematic diagrams of CD20-specific CAR constructs. (B) Representative histogram of CAR expression. Supplemental Figure S2. (A) Gating strategy for analyzing proliferation of CFSE-stained T cells. (B) Histograms of CFSE dilution of CD3+ T cells corresponding to the experiment in Figure 2. Supplemental Figure S3. Proliferation and cytokine secretion of 1F5-28-BB-z CAR. Supplemental Figure S4. Proliferation, cytokine secretion, and cytotoxicity of CAR T cells with fully human anti-CD20 scFv. Supplemental Figure S5. CD20 expression of K80-20low, K80-20med, K80-20high as determined by flow cytometry. Supplemental Figure S6. The absolute cytokine concentrations from T cell supernatants from the experiment in Figure 4 are shown. Supplemental Figure S7. Rituximab-refractory Raji-ffLuc have the same CD20 expression as parental Raji-ffLuc cells. Supplemental Figure S8. (A) Individual mouse bioluminescent tumor burden traces over time corresponding to Figure 5. (B) Representative mouse bioluminescence images. Supplemental Figure S9. Presence of circulating T cells in mice. Supplemental Figure S10. Ofatumumab blocks antigen binding of Ab used to derive CAR scFv.


Damon Runyon-Pfizer Clinical Investigator

Giuliani Family Foundation


NIH/NCI Cancer Center Support


Lymphoma Research Foundation



CD20 is an attractive immunotherapy target for B-cell non-Hodgkin lymphomas, and adoptive transfer of T cells genetically modified to express a chimeric antigen receptor (CAR) targeting CD20 is a promising strategy. A theoretical limitation is that residual serum rituximab might block CAR binding to CD20 and thereby impede T cell–mediated anti-lymphoma responses. The activity of CD20 CAR-modified T cells in the presence of various concentrations of rituximab was tested in vitro and in vivo. CAR-binding sites on CD20+ tumor cells were blocked by rituximab in a dose-dependent fashion, although at 37°C blockade was incomplete at concentrations up to 200 μg/mL. T cells with CD20 CARs also exhibited modest dose-dependent reductions in cytokine secretion and cytotoxicity, but not proliferation, against lymphoma cell lines. At rituximab concentrations of 100 μg/mL, CAR T cells retained ≥50% of baseline activity against targets with high CD20 expression, but were more strongly inhibited when target cells expressed low CD20. In a murine xenograft model using a rituximab-refractory lymphoma cell line, rituximab did not impair CAR T-cell activity, and tumors were eradicated in >85% of mice. Clinical residual rituximab serum concentrations were measured in 103 lymphoma patients after rituximab therapy, with the median level found to be only 38 μg/mL (interquartile range, 19–72 μg/mL). Thus, despite modest functional impairment in vitro, the in vivo activity of CD20-targeted CAR T cells remains intact at clinically relevant levels of rituximab, making use of these T cells clinically feasible. Cancer Immunol Res; 4(6); 509–19. ©2016 AACR.See related Spotlight by Sadelain, p. 473.

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